The present invention relates to a method for identifying a ligand capable of selectively modulating a functional cascade involving a target molecule or target, and also to uses thereof for high-throughput screening of molecules of interest, in particular therapeutic interest (medicament).
Recent advances in the fields of genomics and proteomics, which have led to the identification of a considerable number of new potential therapeutic targets, have opened up very interesting perspectives in the pharmaceutical field.
However, the discovery of new medicaments involves developing methods for high-throughput screening of libraries of molecules which are effective, i.e. which make it possible to isolate, from a restricted number of selected molecules (hits), molecules which are active in vivo on the pathology intended to be treated (leads).
In fact, functional tests (in vitro in cell culture or in vivo in an appropriate animal model) which make it possible to verify the activity of the hits selected during the screening are not generally adaptable to high-throughput.
Since most eukaryotic proteins are multifunctional and involved in several functional cascades, the molecules isolated must be capable of specifically modulating the property of interest of the target without affecting its other activities, the modulation of which could have detrimental effects for the cell. Consequently, the screening must be highly specific in order to restrict to a maximum the number of hits identified, so that only a small number of them have to be tested in functional tests in vivo.
These screening methods must also be easy to carry out and be applicable to any target.
With this aim, screening methods based on the identification of molecules (mimotopes) that mimic the interaction between the target and a peptide or an oligonucleotide (aptamer) have been proposed.
Screening methods using aptamers and/or peptides are illustrated in Lapan et al., Expert Opin. Ther. Targets, 2002, 6, 507-516; Green et al., BioTechniques, 2001, 30, 1094-1110; Burgstaller et al., Drug Discovery Today, 2002, 7, 1221-1228; PCT international applications WO 98/19162 and WO 03/059943 and American U.S. Pat. No. 6,329,145.
In a first step, aptamers/peptides capable of binding to the target are selected in vitro, and then their modulatory activity with respect to the target is verified by means of a functional test in vitro in an appropriate cell system or in vivo in an appropriate animal model.
In a second step, the aptamers/peptides that have a modulatory activity on the target are used in competitive binding assays for identifying hits capable of displacing the existing binding between the aptamer/peptide and the target. The modulatory activity of the hits with respect to the target is subsequently verified by means of a functional test in vitro in cell culture or in vivo in an appropriate animal model, so as to isolate “lead” molecules.
These methods have been validated with targets such as the BB isoform of human platelet-derived growth factor (PDGF BB) and the human immunodeficiency virus (HIV) Rev protein.
The inventors have given themselves the aim of developing alternative screening methods. They have shown that substitution of the modulatory aptamers or peptides with modulatory antibodies or antibody fragments, in the screening methods as described in the prior art, makes it possible to identify molecules capable of selectively modulating a functional cascade involving a target (metabolic cascade, activation cascade, signaling cascade, etc.).
Antibodies have the advantage of recognizing any region of a protein, specifically and with a high affinity (of the order of 1 nM). In particular, they can recognize the surface sites of this protein and are therefore very advantageous for specifically inhibiting the regions of proteins involved in functional cascades via protein-protein interactions; it has in particular been shown that antibody fragments (scFv) directed against the SH2 domains of the Syk protein tyrosine kinase are capable of specifically inhibiting, in vivo, the phospholipase C-γ2 pathway (PLC-γ2; Dauvilliers et al., J. Immunol., 2002, 169, 2274-2283). Consequently, the mimotopes isolated by means of a screening method using antibodies or antibody fragments should have the same properties, in particular in terms of affinity/specificity for the target.
For example, the inventors have screened a library of 3000 molecules using an antibody directed against the SH2 domains of the Syk protein tyrosine kinase and capable of inhibiting the PLCγ-2 pathway; among the 10 ligands of the Syk protein that they selected, at least one was an inhibitory molecule in vivo, acting on the PLC-γ2 pathway, without impairing the Ras pathway, which is also dependent on the Syk protein.
Conversely, it has not been demonstrated that aptamers and peptides are capable of recognizing any surface epitope of an antigen. By virtue of their small size, peptides and aptamers are more suitable for interacting in the cavities of targets rather than at the surface, which is favorable for inhibiting an enzymatic activity but not very advantageous for inhibiting functional cascades.
Moreover, a certain number of recombinant monoclonal antibodies are used in therapeutic treatments. Mention may, for example, be made of cetuximab (anti-EGFR), rituximab (anti-CD20), infliximab (anti-TNF), etc. These antibodies are either chimeric antibodies, most commonly mouse/human, or humanized antibodies. These antibodies are difficult to produce and are very expensive. The screening method proposed by the inventors makes it possible to substitute these antibodies with chemical molecules having the same inhibitory properties, that are easier to produce at a much lower cost (by at least a factor of 10).